1. Field of the Invention
This invention relates to a method and apparatus for producing a probe carrier. More specifically, the invention relates to a method and apparatus for producing a probe carrier which comprises probes fixed in a two-dimensional array arrangement onto a carrier.
2. Description of the Related Art
In analyzing the base sequence of gene DNA, or in making genetic diagnosis simultaneously for many items with high reliability, it is necessary to screen DNA having the desired base sequence with the use of plural types of probes. DNA microchips have attracted attention as means of providing the plural types of probes used in this screening procedure. High throughput screening or combinatorial chemistry for drugs, etc. also requires that many solutions of candidate proteins or drugs (for example, 96 types, 384 types or 1,536 types) be arranged, and subjected to orderly screening. For these purposes, methods for arranging many types of drugs, screening techniques automated in the arranged state, dedicated devices, and software for controlling a series of screening operations or processing the results statistically are under development.
These parallel screening operations basically use so-called probe carriers, each of which comprises many known probes, as screening means, arranged for a substance to be evaluated, thereby detecting the presence or absence of actions on or reactions with the probes under the same conditions. Generally, what actions on or reactions with probes should be utilized is determined beforehand. Thus, the probes loaded on one probe carrier belong to one type of substance, if classified roughly, such as a group of DNA probes with different base sequences. That is, substances utilized as one group of probes include, for example, DNA's, proteins, and synthetic chemicals (drugs). In many cases, a probe carrier comprising plural types of probes forming a group is used. Depending on the nature of the screening operation, an arrayed form in which DNA's having the same base sequence, proteins having the same amino acid sequence, or the same chemicals are arranged at many points may be used as probes. Such an array is used mainly for drug screening.
Specifically, a probe carrier comprising plural types of probes forming a group often takes a form in which plural types constituting a group of DNA's having different base sequences, a group of proteins having different amino acid sequences, or a group of different chemicals are arranged in an array on a carrier or the like according to a predetermined order of arrangement. Of these probe carriers, the DNA probe carrier is used in analyzing the base sequence of gene DNA or in conducting simultaneous, highly reliable genetic diagnosis of many items.
One of the problems with the probe carrier comprising plural types of probes forming a group is how to place as many types of probes as possible, e.g., DNA probes having many types of base sequences, on one carrier. In other words, the problem is how to arrange the probes in an arrayed form at a high density.
As one method for fixing plural types of probes in an arrayed form on a carrier, there can be named a method described in U.S. Pat. No. 5,424,186, the method for preparing DNA probes having different base sequences in an arrayed form by a sequential elongation reaction of DNA on a solid phase carrier with the use of photodegradable protective groups and photolithography. The use of this method makes it possible to produce, for example, a DNA probe carrier loaded with more than 10,000 types, per cm2, of DNA's different in sequence. With this method, when DNA is to be synthesized by a sequential elongation reaction, a photolithography step is performed using dedicated photomasks for four types of bases (A, T, C, G) to elongate any of the bases selectively at a predetermined location in the array, thereby synthesizing plural types of DNA's having desired base sequences in a predetermined arrangement on a carrier. As a DNA strand lengthens, the costs and the amount of time associated with its production increase. In addition, the efficiency of nucleotide synthesis at each elongation stage is not 100%, so that the proportion of DNA's undergoing a deficiency in the designed base sequence is not low. Furthermore, the use of the photodegradable protective group during synthesis results in a low efficiency of synthesis, in comparison with the use of an ordinary acid-degradable protective group. Thus, an array obtained finally shows a low proportion of DNA's having the designed base sequences.
Moreover, the product directly synthesized on the solid phase carrier is used in an unchanged form. Thus, it is, of course, impossible to remove DNA having a defective base sequence from DNA's having the designed base sequences by purification and separation. Another possible problem is that the base sequences of DNA's synthesized on the carrier cannot be confirmed in the resulting array.
This problem means the following. Assume that a small elongation of a predetermined base takes place during a certain elongation stage, because of a mistake in the process or the like, leading to the appearance of a completely defective product. Screening using this defective probe carrier gives erroneous results, but this cannot at all be prevented. This inability to confirm the base sequence is a major and essential problem with the above-described method.
Another method proposed for producing a probe carrier comprises synthesizing and purifying DNA's for probes beforehand, confirming their base lengths if desired, and supplying the respective DNA's onto a carrier by a device such as a microdispenser. PCT International Patent Application Publication No. WO 95/35505 describes a method of supplying DNA's onto a membrane by use of a capillary. The application of this method, in principle, enables an array of about 1,000 DNA's/cm2 to be produced. Basically, this method involves supplying a probe solution to a predetermined position on a carrier by a single capillary-shaped dispense device for each probe, and repeating this procedure, thereby producing a probe carrier. No problem is posed if dedicated capillaries are used for the respective probes. However, if a small number of capillaries is used to perform the same procedure, the capillaries need to be washed thoroughly in changing the types of the probes in order to prevent mutual contamination. The position of the supply should also be controlled for each procedure. Thus, the method is not said to be suitable for producing an array having many types of probes arranged at a high density. Besides, the supply of the probe solution to the carrier is carried out by tapping the front end of the capillary on the carrier. Thus, neither reproducibility nor reliability is complete.
As another method, a proposal has been made for a method in which when solid phase synthesis of DNA is performed on a carrier, a solution of a substance necessary for synthesis is supplied onto the carrier by the ink-jet method at each elongation stage. For example, European Patent Publication No. EP 0 703 825 B1 describes a method for solid phase synthesis of plural types of DNA's having predetermined base sequences by supplying nucleotide monomers and activators, which are utilized in solid phase synthesis of DNA, by different piezo jet nozzles. The supply (coating) by this ink-jet method, as contrasted with the supply (coating) of a solution using the capillary, is high in reliability, as seen from reproducibility of the amount of supply, and permits microfabrication of the structure of the nozzle. Thus, this method has features suitable for attaining a high density probe carrier. However, this method also basically utilizes a sequential elongation reaction of DNA on the carrier. Thus, it still has problems, such as the aforementioned major problem with the method described in U.S. Pat. No. 5,424,186, i.e., the base sequences of DNA's synthesized on the carrier cannot be confirmed. This method eliminates the tiresome procedure of performing a photolithography step using a dedicated mask at each elongation stage, but remains slightly problematic in terms of a requirement that the predetermined probes be fixed at respective points, which requirement is indispensable to the probe carrier. The aforementioned publication EP 0 703 825 B1 describes only a method using a plurality of individually formed piezo-jet nozzles. The method using the small number of the nozzles, like the aforementioned method using capillaries, is not necessarily suitable for producing a high density probe carrier.
Japanese Patent Application Laid-open No. 11-187900 (1999) discloses a method for forming spots containing probes on a solid phase by adhering liquids containing the probes, as droplets, to the solid phase by a thermal ink jet head. However, the ink jet head used is an ordinary printer head, and thus does not have an optimal structure for producing a probe carrier. Detailed reasons for this will be described below.
The conventional ink jet head was developed for printing characters or images. Thus, the solution used for it is ink of a single color (black) in the case of monochromatic (generally black) printing, or generally comprises inks of three primary colors, i.e., yellow (Y), cyan (C) and magenta (M), in the case of color printing. In color printing, variable density black or Y, M and C inks may be used where necessary, but more than ten types of inks, at most, are not used.
A large amount of ink is used for printing on a paper face. Thus, the conventional head for ink jet printing is equipped with a full capacity tank (reservoir) to be filled with ink, a channel for guiding the ink to a nozzle, and the nozzle for ejecting the ink.
A liquid ejection head for production of a probe carrier, on the other hand, is required to eject as many types of liquids as possible, as explained earlier. If the head has a plurality of nozzles, the head desirably has the same number of solution reservoirs as the number of the plural nozzles, the solution reservoirs corresponding one-to-one to the plurality of nozzles.
With a liquid ejection device for production of a probe carrier, moreover, the consumption of the liquid is small compared with printing on paper, so that the reservoir with a relatively small capacity is sufficient.
With the conventional ordinary head for ink jet printing, furthermore, desired ink needs to be ejected at a desired position on the paper face in order to form characters or images. Thus, the head is configured to be capable of selecting respective nozzles independently with arbitrary timing. Powered transistors and logical circuits, necessary for ejecting ink (liquid) from the desired nozzles, may be provided outside or inside the head.
Ink jet systems are classified into a thermal jet system for ejecting the liquid by thermal energy generated by a heater and a piezo-ink jet system for ejecting the liquid by deformation of a piezoelectric element caused when a voltage is applied to the piezoelectric element. Of these systems, the thermal jet system is simple in structure as compared with the piezo-ink jet system and is suitable for a downsized head and a multi-nozzle head.
As described above, the method of producing a probe carrier by means of a liquid ejection device is excellent in that minute amounts of probe solutions to be imparted are arranged at a high density on a solid phase carrier. The minimum amount of the liquid ejected at a time from an ejection orifice of the liquid ejection device can be decreased to the range of 0.1 pl to 50 pl. Even when many wells are placed on the carrier and minute amounts of the probe solutions are supplied into the respective wells, the sizes of the wells can be made small to achieve an even higher density.